The long term objective of this proposal is to elucidate the molecular basis by which collectins in the lung provide the first challenge to airborne pathogens in host defense. The surfactant specific collectins SP-A and SP-D, through their interactions with lipopolysaccharides(LPS), are responsible in part for this activity. With the technological expertise in novel vibrational spectroscopic appraoches developed in this lab, we propose two specific aims to characterize the interaction of both proteins with physiologocal LPS derivatives. With the expertise of the other investigators in the program project, we will have access to a large number of genetic variants of these proteins, permitting us to pinpoint the primary interaction sites in a variety of biologically relevant physical preparations. Two specific aims are proposed. First, since both collectins recognize carbohydrate and other polar ligand sites, we will determine those collectin structural factors important for binding to LPS and its variants in Langmuir films (i.e. monolayers) at the air/water interface using a unique vibrational spectrscopy experiment (Infrared Reflection Absorption Spectroscopy- IRRAS) that extracts molecular structural and orientational information from the monolayer constituents. These films mimic polar regions of bacterial Gram negative outer membrane monolayers. The integrating hypothesis for this Aim is that at the level of molecular structure, the interaction between collectins and LPS depends on particular elements of protein structure and emphasizes specific regions of the collectin-carbohydrate recognition domain. Raman microcrystallography complements IRRAS and provides very specific structural information about protein side chains involved in the initial recognition event. In the second Aim, we will complement studies of protein structural changes with IR experiments that track structural changes both in the acyl chains and in the polar regions of the LPS derivatives. We will monitor collectin/LPS interaction under conditions where Langmuir films are not the only reasonable experimental paradigm. Physical states of lipid and lipid/protein complexes from which structural information will be acquired include vesicles, monolayers, supported oriented multibilayers, and micelles. RELEVANCE (See instructions): The collectins play a vital role in host defense against microorganisms, although little is known of the molecular basis for microbial recognition. Following the planned novel vibrational spectroscopic studies, a more detailed understanding of this mechanism of host defense will emerge. Since threats to personal security are currently presumed significant, understanding the recognition mechanisms are important for effective design of agents for therapeutic intervention